Proteins are primary components of all living matter. They play crucial roles in virtually all biological processes. They may be classified according to biological function as the enzymes, hormones, the immunoglobulins or antibodies, the clotting proteins and the toxins. Other proteins that have less intense biological activity are the transport proteins, the storage proteins, the contractile and structure proteins. Many of these essential proteins are found in the circulating plasma. Thus, a simple and reliable method of labeling these substances with a suitable radionuclide which preserve their physiobiological properties offers unlimited potential in biomedical applications.
Despite the initial optimism over possible clinical application of radiolabeled proteins, the usefulness of these agents has been limited to mainly in in vitro clinical assays. Radioimmuno assays (RIA) using .sup.125 I-labeled antibodies has found a wide range of clinical applications in medicine. The versatility, sensitivity and accuracy of these RIA techniques are well documented in the medical literature. Attempts to use radiolabeled plasma proteins as scintigraphic imaging agents in Nuclear Medicine have been disappointing. There are only few radiolabeled plasma proteins commercially available for scintigraphic imaging purposes. These include .sup.131 I-labeled or .sup.99m Tc-labeled human serum albumin(HSA). Technetium-99m HSA is widely used in cardiology as a blood pool imaging agent, whereas, .sup.131 I-HSA has limited application in cisternography as an alternative to .sup.111 In-DTPA. HSA labeled with .sup.125 I has found useful application in blood volume determination as an adjunct to other clinical procedures. Although recent introduction of .sup.125 I-human fibrinogen has offered the clinicians a more sensitive means of detecting deep vein thrombosis, it is not a scintigraphic imaging agent. Because of the low energy gamma photon flux of .sup.125 I, the usefulness of radioiodinated fibrinogen is limited to surface counting technique on the lower extremities. A more wide spread use of radiolabeled plasma proteins in Nuclear Medicine has been severly restricted because of: (1) in vivo instability of radioiodinated proteins; (2) undesirable isotopic characteristics of the radionuclides used in the labeling process; (3) protein denaturation caused by the current labeling techniques; (4) potential danger of antigenic reaction and high risk of hepatitis transmission from radiolabeled exogenous plasma proteins.
Several critical parameters must be met before radiolabeled protein substances can be used as scintigraphic imaging agents. Among these are: (1) they must be stable and biologically active in the body; (2) they must be highly selective or specific for the targeted organ or lesion; (3) the radionuclide must be firmly bound to the protein ligand and stable for the duration of the study; (4) the radionuclide must have favorable isotopic characteristics that are compatible with conventional imaging equipments and finally, (5) these radiolabeled proteins must not be toxic or antigenic to human subjects.
Protein denaturation and complete loss of biological properties are the primary concern in protein labeling chemistry. Various techniques of labeling plasma proteins with .sup.125 I, .sup.123 I or .sup.131 I have been reported in the literature. The most commonly used chemical means is radioiodination of the protein in the presence of Chloromine-T or iodine monochloride. The labeling yields, however, is low and varies from 50-70%. In order to be clinically useful as radiopharmaceuticals, the desired labeled proteins must undergo a long and tedious separation and purification process. Radioiodinated proteins are unstable in vivo. The radionuclide is rapidly detached from the protein ligand caused by dehalogenation as evident by unusually high tissue background radioactivity observed in the scintigrams. Furthermore, there is increasing evidence that alternation of the protein molecular structure occurs during radioiodination.
Technetium-99m labeled human plasma proteins have not found wide acceptance in Nuclear Medicine. Although several plasma proteins such as HSA, fibrinogen, antibodies or antibody fragments have been labeled with .sup.99m Tc by chemical means, only .sup.99m Tc-HSA has been approved for human use. One major problem is that plasma proteins labeled by the current techniques either by Sn-acid reduction process at pH of less than 2 (U.S. Pat. No. 4,042,676 to Molinski et al, U.S. Pat. No. 4,094,965 to Layne and U.S. Pat. No. 4,311,688 to Rhodes) or by Sn-basic reduction method at pH 11.6 (U.S. Pat. No. 4,057,617 to Abramovici) are completely denatured with significant loss of physiobiologicaly properties. This renders them unsuitable for biomedical applications. HSA labeled with .sup.99m Tc, for example, does not have the same native biological property as that of the unlabeled serum albumin. Thus, its usefulness has been limited to cardiac studies as a blood pool scintigraphic imaging agent. A second major obstacle which prevents a wider use of radiolabled plasma proteins is the high risk of antigenic reaction due to denatured protein byproducts. To resolve these problems, the present inventor has developed a simple chemical method of labeling plasma proteins with .sup.99m Tc under physiological condition (U.S. Pat. No. 4,293,537 to Wong). The basic labeling methodology involves the production of a stable and chemically active .sup.99m Tc-(Sn)citrate complex species in neutral medium prior to the addition of the protein. The actual binding of .sup.99m Tc to the protein ligand occurs at physilogic pH 7.4 condition, thus avoiding harsh treatment of the protein and preserving its native biological properties. (Wong DW, et al, J. Nucl. Med. 20:967, 1979 and Wong DW, et al, J. Nucl. Med. 23: 229, 1982).
Technetium-99m labeled plasma proteins which retain their natural biological properties after labeling are ideal scintigraphic imaging agents. Essentially, these radiolabeled protein substance will actively participate in the physiobiological processes in the body. Tc-99m labeled autologous antimicrobial or anti-tumor antibodies, for examples, are immunologically active against specific antigens. This provides a simple, unique and highly specific means of detecting infectious lesions or neoplasms. Similarly, diseases such as venous and arterial thrombosis, pulmonary or cerebral embolisms, myocardial infarction and tumors can be diagnosed using .sup.99m Tc-labeled autologous human fibrinogen. One major disadvantage of .sup.99m Tc-labeled compound is the isotopic characteristics of the radionuclide itself. Although .sup.99m Tc has an ideal 140 KeV gamma photon flux compatible with existing scintigraphic imaging equipments, but it has a relatively short physical half-life of only 6 hours. This renders .sup.99m Tc-based radiopharmaceuticals unsuitable for imaging studies that require observation period of more than 6 hours. A 24 hours delayed imaging study, for example, requires a dose of 25-50 mCi of the radiopharmaceutical. For imaging studies in excess of 24 hours, repeated injections are needed. However, the usefulness of radiolabeled plasma proteins can be extended considerably if they are labeled with a longer physical half-life radionuclide such as Indium-111(.sup.111 In) or Gallium-67 (.sup.67 Ga) which has similar gamma photon energy.
Currently, there are only two medical useful radionuclides of Indium. These are .sup.111 In and .sup.113m In. Of these, .sup.111 In possess the most ideal radioisotopic characteristics for scintigraphic imaging procedures. Indium-111 has a 2.83 days half-life and produces two gamma photons (173 and 247 KeV, respectively) per disintegration. Both of these are in a useful energy range for imaging with standard Nuclear Medicine equipments. The resultant 183% photon production per disintegration produces a very high photon flux per mCi administered. Because of its longer half-life, .sup.111 In-based radiopharmaceuticals are ideally suited for imaging studies that require observation period in days or weeks. Optimal delayed images can be obtained with a single injection of a small dose of the radiolabeled compound and yet produces minimal amount of radiation health hazard to patient. Indium-113m is also a pure gamma emitter which emits a 301 KeV gamma photon. However, it has a very short half-life of 1.65 hours. Indium-113m based radiopharmaceuticals are not suitable for imaging studies that require observation period of more than 3 hours.
Several indirect methods of labeling plasma proteins with .sup.111 In have been reported in the literature(Burchiel and Rhode, Radioimmunoimaging and Radioimmunotherapy, Elseier Science Publishing Co. N.Y., 1983). The most common technique requires the use of a bifunctional chelate, a compound which posses two binding sites; one site for chelating polyvalent metallic ion and a second site for coupling to the protein ligand. Basically, the chelate such ad EDTA(ethylenediamine tetraacetic acid) or DTPA(diethylenetriamine pentaacetic acid) is first converted to the acid anhydride form by reflux reaction with trifluoroacetic acid and thionyl chloride. A precipitate of EDTA-anhydride or DTPA-anhydride is formed and purified with anhydrous ether. The protein substance is then conjugated or coupled to the EDTA-anhydride or DTPA-anhydride by incubating the reaction mixture at 4.degree. C. for 24 hours. The reaction mixture is then dialyzed at 4.degree. C. for another 24 hours to remove chemical impurities, purified again by column chromatography and stored in a frozen state until needed for labeling. During labeling procedure, .sup.111 InCl.sub.3 is added and is chelated to the EDTA- or DTPA-conjugated protein in an acidic pH 4 medium following a 2 hours room temperature incubation period. The radiolabeled product is again purified by gel column filtration to obtain the pure radiolabeled protein and to remove radiochemical impurities such as free or unbound .sup.111 In, .sup.111 In-DTPA and insoluble .sup.111 In-hydroxide colloids.
A closer examination of the bifunctional chelation labeling process reveals many flaws. This labeling process is extremely complicated, tedious and time consuming accompanying with very poor labeling yields. A binding or labeling efficiency ranging from 10-70% prior to purification process indicate that the bifunctional chelation technique is neither reliable nor reproducible. The primary problem is due to chemical decomposition of the cyclic anhydride caused by hydrolysis in the presence of air and moisture even at refrigeration temperature. Hydrolysis occurs more rapidly at room temperature causing complete decomposition of the cyclic anhydride. Thus, the coupling reaction of protein to cyclic anhydride must be carried out at 4.degree. C. to avoid hydrolysis. The presence of hydrolyzed unconjugated DTPA or EDTA will compete with the protein ligand for the radionuclide resulting in reduced labeling yeilds. Since the labeling process takes place in room temperature at acidic pH condition, hydrolysis of the cyclic anhydride and denaturation of the protein will occur. Although proteins such as fibrinogen and antibodies labeled by this process have claimed to be biologically active, experimental evidence indicate that as much as 50% of the biological properties of the native protein is lost after labeling process (Scheinberg D. A., et al, Science 215: 1511, 1982 and Layne W. W., et al, J. Nucl. Med. 23: 627, 1982). The entire labeling process from the production of EDTA- or DTPA-anhydride protein conjugate to the actual coupling reaction with the radionuclide requires a minimum of 3-4 days. Extensive purification steps are needed to obtain pure radiolabeled protein. This increases the risk of microorganism and pyrogen contamination. The bifunctional chelation labeling process is neither simple, practical nor suitable for preparing pharmaceutically acceptable radiolabeled protein substances.
The present invention is the result of extensive investigation of the .sup.99m Tc physiologic chemical labeling process developed by the present inventor. Experimental data confirm that the (Sn)citrate chemical species are capable of forming complex bimetallic chemical species with polyvalent metallic ions such as .sup.99m Tc or .sup.111 In. These bimetallic complex species posses high protein binding property. They are also chemically active and stable at neutral pH 6-8 medium a condition is ideally suited for labeling protein substances.
The present invention offers many obvious advantages over the bifunctional chelation process for labeling plasma proteins with the radionuclides of Indium. Among these are: (1) the labeling process is a simple and a direct chemical method of incorporating the radionuclide to the protein ligand; (2) the labeling process is mild and without the use of toxic chemical reagents; (3) since plasma proteins are labeled under optimal physiologic pH 7.4 condition, their physiobiological properties are preserved. There is no evidence of protein denaturation or decomposition caused by the present labeling process; (4) the labeling yields is greater than 98% with excellent reliability and reproducibility; (5) the radionuclide is firmly bound to the protein ligand and free from radiochemical impurities; (6) the radiolabeled proteins are biochemically active and stable in excess 3 months period when properly stored at 2.degree.-8.degree. C.; (7) the radiolabeled protein is ready for use after labeling without the need of any purification steps, and finally, (8) the entire labeling process requires less than 1 hour of time and uses only few simple non-toxic chemicals. The present labeling process can be converted into an instant non-radioactive labeling reagent kit to facilitate in-house preparation of exogenous or autologous radiolabeled plasma protein injections.